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Abstract

Combined non-linear imaging techniques were used to deeply image human ex-vivo fresh biopsies of bladder as well as to discriminate between healthy bladder mucosa and carcinoma in situ. Morphological examination by two-photon excited fluorescence and second-harmonic generation has shown a good agreement with corresponding common routine histology performed on the same samples. Tumor cells appeared slightly different in shape and with a smaller cellular-to-nuclear dimension ratio with respect to corresponding normal cells. Further differences between the two tissue types were found in both spectral emission and fluorescence lifetime distribution by performing temporal- and spectral- resolved analysis of fluorescence. This method may represent a promising tool to be used in a multi-photon endoscope, in a confocal endoscope or in a spectroscopic probe for in-vivo optical diagnosis of bladder cancer.

Histogram distribution of the cellular-to-nuclear area ratio (d) for CIS (in red) and for HM (in black), calculated by summing over images acquired in a 20-30 μm, on 5 samples of CIS and 5 samples of HM and over 10 cells per sample.

Two-photon emission spectra for CIS (red) and for HM (black), measured by summing the detected photon number over a region of 20 μm × 20 μm at a depth of 50-60 μm, and by averaging on 5 samples of CIS and 5 samples of HM, for 740 nm excitation (a), and for 890 nm excitation (b). Two examples of SAAID maps (c), calculated using Eq. (1), for both CIS and HM presented in a color-coded scale. Scale bars: 5 μm. SAAID distribution for CIS (red) and for HM (black) with the color-coded scale used in SAAID mapping superimposed (d), obtained by summing the calculated SAAID index over a region of 20 μm × 20 μm, and after averaging on 5 samples of CIS and 5 samples of HM.